Non-replicating paramyxoviridae virus vector

Abstract

The present inventors succeeded in producing non-replicating SeV vectors whose genomic RNAs lack all genes for the NP, P, and L proteins, which are RNP-constituting proteins. The present inventors confirmed that the NP/P/L-deficient SeV vectors carrying a marker gene such as GFP provide high productivity, and high transfer and expression efficiencies of foreign genes (high MOI infection is essential for achieving high expression levels). By lacking the L gene or two or more of the NP, P, and L genes, the vectors of the present invention enable lowering the level of virus-derived proteins expressed in host cells, thereby reducing the immunogenicity upon in vivo administration.

1 . A vector that comprises a complex comprising:
 (a) a negative single-stranded RNA derived from a Paramyxoviridae virus, wherein said negative single-stranded RNA has been modified so as not to express one or more proteins, selected from the NP, P, and L proteins, including at least the L protein, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) the NP, P, and L proteins.   
     
     
         2 . A vector that comprises a complex comprising:
 (a) a negative single-stranded RNA derived from a Paramyxoviridae virus, wherein said negative single-stranded RNA has been modified so as not to express multiple proteins selected from the NP, P, and L proteins, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) the NP, P, and L proteins.   
     
     
         3 . The vector of  claim 1  or  2 , wherein the Paramyxoviridae virus-derived negative single-stranded RNA has been modified so as not to express the NP, P, and L proteins. 
     
     
         4 . The vector of any one of  claims 1  to  3 , wherein the Paramyxoviridae virus-derived negative single-stranded RNA expresses at least one of the Paramyxoviridae virus envelope proteins. 
     
     
         5 . The vector of  claim 4 , wherein the Paramyxoviridae virus-derived negative single-stranded RNA has been modified so as to express the M, F, and HN proteins, and not to express the NP, P, and L proteins. 
     
     
         6 . The vector of any one of  claims 1  to  5 , wherein the negative single-stranded RNA is derived from Sendai virus. 
     
     
         7 . The vector of any one of  claims 1  to  6 , wherein the negative single-stranded RNA further carries a foreign gene. 
     
     
         8 . A DNA encoding a negative single-stranded RNA comprised in the vector of any one of  claims 1  to  7 , or the complementary strand thereof. 
     
     
         9 . A method for producing the vector of  claim 1 , wherein the method comprises the steps of:
 (a) introducing a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, into cells capable of expressing the NP, P, and L proteins,   wherein said negative single-stranded RNA has been modified so as not to express one or more proteins, including at least the L protein selected from the NP, P, and L proteins, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         10 . A method for producing the vector of  claim 2 , wherein the method comprises the steps of
 (a) introducing a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, into cells capable of expressing the NP, P, and L proteins,   wherein said negative single-stranded RNA has been modified so as not to express multiple proteins selected from the NP, P, and L proteins; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         11 . The production method of  claim 9  or  10 , wherein the cells capable of expressing the NP, P, and L proteins can further express a protein having the function of promoting formation and release of viral particles. 
     
     
         12 . The method of  claim 11 , wherein the protein having the function of promoting formation and release of viral particles is the C protein. 
     
     
         13 . A method for producing the vector of  claim 1 , wherein the method comprises the steps of:
 (a) introducing a vector expressing the NP protein, a vector expressing the P protein, a vector expressing the L protein, and a vector into which a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, is inserted, into cells capable of expressing the vectors,   wherein said negative single-stranded RNA has been modified so as not to express one or more proteins, including at least the L protein selected from the NP, P, and L proteins, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         14 . A method for producing the vector of  claim 2 , wherein the method comprises the steps of:
 (a) introducing a vector expressing the NP protein, a vector expressing the P protein, a vector expressing the L protein, and a vector into which a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, is inserted, into cells,   wherein said negative single-stranded RNA has been modified so as not to express multiple proteins selected from the NP, P, and L proteins; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         15 . A method for producing the vector of  claim 1 , wherein the method comprises the steps of:
 (a) introducing a complex comprising a paramyxovirus-derived negative single-stranded RNA and proteins that bind to said negative single-stranded RNA, into cells expressing the NP, P, L proteins and an envelope protein,   wherein said negative single-stranded RNA has been modified so as not to express one or more proteins, including at least the L protein selected from the NP, P, and L proteins, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         16 . A method for producing the vector of  claim 2 , wherein the method comprises the steps of:
 (a) introducing a complex comprising a paramyxovirus-derived negative single-stranded RNA and proteins that bind to said negative single-stranded RNA, into cells expressing the NP, P, L proteins and an envelop protein,   wherein said negative single-stranded RNA has been modified so as not to express multiple proteins selected from the NP, P, and L proteins, which bind to a Paramyxoviridae virus negative single-stranded RNA; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         17 . A method for producing a vector for expressing a foreign gene in target cells, wherein the method comprises the steps of:
 (a) introducing a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, into cells capable of expressing the NP, P, and L proteins,   wherein said negative single-stranded RNA has been modified so as not to express one or more proteins, including at least the L protein selected from the NP, P, and L proteins, and carries a foreign gene; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         18 . A method for producing a vector for expressing a foreign gene in target cells, wherein the method comprises the steps of:
 (a) introducing a DNA encoding a Paramyxoviridae virus-derived negative single-stranded RNA, or the complementary strand thereof, into cells capable of expressing the NP, P, and L proteins,   wherein said negative single-stranded RNA has been modified so as not to express multiple proteins selected from the NP, P, and L proteins, and carries a foreign gene; and   (b) culturing the cells, and collecting viral particles from the culture supernatant.   
     
     
         19 . A method for expressing a foreign gene in target cells, wherein the method comprises the steps of:
 (a) producing the vector of  claim 7 ; and   (b) introducing the vector of  claim 7  into target cells.